Dihydrolipoamide dehydrogenase (E3) is a common component of the alpha- ketoacid dehydrogenase complexes which are involved in the oxidative decarboxylation of pyruvate, alpha-ketoglutarate and the branched-chain alpha-keto acids, and hence plays an important role in intermediary metabolism. Patients with E3 deficiency have developmental delay and severe neurologic disability. The long term goal of this proposal is to better understand the structure-function relationship of human E3 at the protein level and regulation of the human E3 gene in normal and disease states. The availability of a full-length human E3 cDNA clone offers the opportunity to pursue these goals. Four specific aims of this proposal are: (i) to investigate the structure-function relationship of both normal and site-specifically mutated human E3s, (ii) to investigate genomic organization and to characterize the promoter-regulatory region of the human E3 gene, (iii) to identify the molecular mechanism(s) causing E3 deficiency in affected patients, and (iv) to develop appropriate chimeric genes to express the human E3 gene product in E3-deficient skin fibroblasts derived from E3-deficient patients. A3heterologous expression vector containing human E3 cDNA (pOTSV3-E3) will be used to express human E3 (mature form) in E. coli. Using site-directed mutagenesis, specific amino acid residues in the active site and at the site(s) of interaction with the other component(s) of the complex will be altered in human E3 and their structure-function relationship will be analyzed. Using plaque hybridization, genomic clones for the human E3 gene will be isolated and characterized with respect to their organization (including intron/exon junctions, transcription start sites, etc.) and the promoter-regulatory region of the gene. Mutation analysis in E3-deficient patients will be carried out at the levels of E3 protein (Western analysis), mRNA (Northern analysis) and DNA (Southern analysis, polymerase chain reaction generated DNA sequencing, analysis of genomic DNA fragments). Retroviral vectors will be constructed containing the phosphoenolpyruvate carboxykinase promoter ligated to the E3 structural gene to stably integrate and express human E3 in mammalian cells. Our multifaceted approach to studying the E3 protein and its gene should enhance our understanding of structure-function relationship of E3 protein and the regulation of the E3 gene. Techniques developed for this study will have wider application to other metabolic birth defects.